The goal of this project is to determine how Matrix Gla protein (MGP) acts as an inhibitor of calcification. The working hypothesis is that MGP interacts directly with nascent crystal nuclei to inhibit calcification of arteries and regulate calcification in bone. The first aim is to understand how MGP inhibits calcification using a supersaturated calcium and phosphate system that, in the absence of MGP, spontaneously forms apatitelike mineral. MGP will be added to a calcium and phosphate buffer system to determine how it may affect the formation of a bone-like mineral precipitate. The second aim is to understand how MGP prevents the serum-initiated calcification of arteries and demineralized bone. To address mechanistically how MGP functions in both of these systems we plan to test the activity of modified forms of MGP prepared by specific chemical modifications and by site-directed mutagenesis and to characterize the complex formed between MGP and nascent crystal nuclei. The third aim is to develop a method to purify the native conformation of MGP using procedures that avoid the acidic conditions and protein denaturants used in previous MGP purifications. [unreadable] [unreadable]